PROJECT SUMMARY GPR55 is an atypical cannabinoid receptor whose endogenous agonist is L-?-lysophosphatidylinositol (LPI). Emerging evidence indicate new roles for GPR55, including modulation of the function of endothelial cells. This project will investigate the role of GPR55 in the endothelial cells of the blood-brain barrier (BBB). BBB has a critical role in neuroprotection by permitting the transport of nutrients to the brain and removal of metabolites, while protecting the brain from potentially toxic or harmful substances. BBB dysfunction occurs in several neurological and neurodegenerative disorders. A better understanding of the factors that modulate BBB permeability is critical for designing effective therapies for neuroprotection. Our central hypothesis is that LPI activation of GPR55 produces a sustained increase in cytosolic Ca2+, mitochondrial Ca2+ overload and increase in reactive oxygen species (ROS), increasing the BBB permeability. We will use a multidisciplinary approach including measurement of cytosolic and mitochondrial Ca2+, cytosolic and mitochondrial ROS levels, and assessment of endothelial BBB permeability in vitro and in vivo. We will interface pharmacological modulation using specific GPR55 ligands with molecular knockdown approaches. The in vitro experiments will be carried out in rat brain microvascular endothelial cells (RBMVEC). The project has two specific aims: Aim 1: To investigate the role of GPR55 in modulation of cytosolic and mitochondrial Ca2+ in brain microvascular endothelial cells. Ca2+ is a second messenger critical for cellular functions; however, a sustained increase in cytosolic Ca2+ concentration produces mitochondrial Ca2+ overload and initiates oxidative stress. We will use imaging techniques to monitor the changes in cytosolic and mitochondrial Ca2+, cytosolic ROS and mitochondrial ROS in response to GPR55 activation in RBMVEC. Aim 2: To investigate the impact of GPR55?s modulation of endothelial cells Ca2+ upon BBB permeability. Endothelial cells of the BBB form a monolayer with low paracellular permeability and high electrical resistance. We will assess the changes in endothelial permeability produced by GPR55 activation in vitro and in vivo. The results of this project will provide essential information of BBB regulation by cannabinoid receptors, while identifying a new potential therapeutic target for neuroprotection.